Introduction

Diplocrepis puniceus (Richardson, 1846) and Trachelochismus pinnulatus (Forster, 1801) are endemic New Zealand species of the family Gobiesocidae. They are common throughout the mid-tide and low tide levels of the rocky shore around New Zealand as is also the clingfish Trachelochismus melobesia (Phillipps, 1927).

Diplocrepis puniceus is a relatively large clingfish with conspicuous coloration, attaining 100mm standard length. The patterns and intensity of its body colour vary considerably in both male and female. In general, however, the dorsal surface is mottled with light and dark shades of green, contrasting with a pale yellow ventral surface. During the breeding season the males have an overall lilac tinge which is possibly a response to the deep purple colour of the egg clusters which they attend.

In T. pinnulatus the dorsal surface is light or dark green with longitudinal brown bands or brown spots and the ventral surface is pale yellow. Trachelochismus melobesia (max. size 30mm s.l.) is similar to, but smaller than T. pinnulatus (max. size 72mm s.l.) and has a reddish-purple patch on the dorsal surface, which is lacking in T. pinnulatus. Diplocrepis puniceus is distinguished from T. pinnulatus and T. melobesia by its large horse-shoe shaped head and distinctive colour. The three are further distinguished by the following fin-ray counts (Briggs, 1955) :

D. puniceus D11 (10-11), A5 (4-5), PI 23 (23-24), C10.

T. pinnulatus D8 (7-9), A6 (5-7), PI 25 (24-26), C12 (11-12).

T. melobesia D10 (9-11), A8 (7-8), PI 23 (22-24), C12.

The present study describes the embryonic development and early larval growth of D. puniceus and T. pinnulatus. Comparable features of T. melobesia are described elsewhere (Ruck, 1971). The life history and general biology of T. pinnulatus were studied by Coakley (1964), and Graham (1939, 1953) has briefly described the egg and prolarval stages of D. puniceus.

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Materials and Methods

On 21.7.70 an egg mass of D. puniceus (identification from description by Graham, 1939) was collected from the western shore of Lyall Bay on the north coast of Cook Strait. This was kept in a rectangular plastic container (30 × 28 × 13cm) at the Island Bay Marine Laboratory. The water was changed daily and kept constantly aerated.

On hatching, prolarvae of D. puniceus were transferred to plastic containers (12 × 15cm) which had been sterilized by exposure to ultraviolet light. The water in each container was replaced daily with filtered and sterilized seawater. No artificial aeration was provided. Artemia ("brine shrimp") nauplii were fed to the larvae as soon as most of the yolk sac had been absorbed. The last larva died on the 15th day after hatching. During the 15 days several larvae were anaesthetised with Sandoz MS 222 and sketches were made.

Larvae of D. puniceus larger than 5.5mm were also obtained from the plankton approximately 100m off-shore at Island Bay and Lyall Bay, using a plankton net of standard conical design with a 57cm diameter opening and a mesh size of 500 microns.

Early in July, 1970, 10 adult T. pinnulatus were collected from the western shore of Lyall Bay, and kept in a glass aquarium (60 × 40 × 25 cm). Stones, broken clay piping and empty Haliotis (paua) shells were placed in the aquarium to provide shelter and areas on which to spawn. The tank was supplied with fresh running seawater and fish were fed chopped fish and beef liver. On 28.7.70 freshly deposited eggs were found on the undersurface of a portion of the clay pipe. These eggs were attended by a male T. pinnulatus (61mm s.l.) which, along with the egg mass, was transferred to a plastic container (30 × 28 × 13cm). The water was changed daily and supplied continuously with air.

The egg masses of both species adhered closely to the substrate and hence eggs required for observation were very difficult to remove without damage to the egg membrane. Limited success was obtained by sliding a sharp scalpel between the egg and the substrate and then pipetting the dislodged egg into a petri dish. Development of the eggs of D. puniceus and T. pinnulatus were studied and sketches of the eggs and larvae were made with a camera lucida. An ocular micrometer was used for measuring eggs and larvae. Measurements of the larvae are those recommended by Hubbs and Lagler (1958:24-26), with the exception of head length, taken here as the distance from the snout to the posterior margin of the otic capsule. In the prolarva the otic capsule provides a more positive point of reference than the operculum. Descriptive terminology of eggs and prolarvae follows that used by Rugh (1948), Balinsky (1965) and New (1966).

Spawning and Development of D. Puniceus

In the Lyall Bay area D. puniceus spawns from early July to late September. The eggs are deposited on the undersurfaces of large stones and are in close contact, forming flat irregularly shaped egg masses which remain covered with water at low tide. Typically the egg masses are relatively large; one kept in the laboratory measured 13 × 5cm and contained approximately 2,400 eggs. Each egg mass contains sub-groups, each with up to 300 eggs, which differ slightly in colour, ranging from deep purple to pale mauve. The deep purple eggs appear crimson when viewed under the binocular microscope. The variation in colour between sub-groups is mainly a result of the progressive depletion of the yolk page 3supply in the eggs of each sub-group as development advances. This suggests that the eggs of adjacent sub-groups within the same mass are deposited at different times. The pale and small-yolked eggs, which show well-developed eyes, are the most advanced in development and are found mainly in the centre of the egg mass. In some sub-groups, however, the well-developed eggs retain deep purple yolks.

The male collected with the egg mass from Lyall Bay remained with the eggs constantly. It did not eat food offered. It swam backwards and forwards across the eggs, maintaining a constant flow of water over the egg mass. This helped to remove detritus from the egg membranes, and made observation of the embryo through the chorion relatively easy. By comparison the chorion of eggs kept separate from the male soon became covered in foreign particles to such a degree that the embryo and yolk within were very difficult to see.

Development of the Egg and Larvae

Most of the eggs within the clusters are laid so close together that they impinge on each other and so deform the chorion (Fig. 1, No. 1). The consequent irregularities in the shape of most eggs make accurate measurement of the egg diameter difficult.

Eggs that make no contact with their neighbours, however, are spherical when viewed dorsally, and have a mean diameter of 1.80mm. The yolk is sub-spherical and in a standard sample of 100 eggs has a mean diameter of 1.30mm. During early development the yolk is dark purple and contains a single large oil globule (mean diameter 0.45mm) and 20-30 smaller ones. The eggs are dorso-ventrally depressed and are attached to the substrate by a flattened adhesive base.

Development to hatching (Fig. 1, Nos. 1-12) takes 24 days at 11.57°C. No eggs were found earlier than the gastrulation stage, and these were estimated to be nearly two days old.

Second day (Fig. 1, No. 1). At this stage the blastodisc has spread halfway around the yolk. Epiboly is not obvious, except for a slight thickening of the germ ring.

Third day (Fig. 1, Nos. 2 & 3). The embryonic shield is well defined and lies deeply notched into the yolk. The blastodisc continues to expand over the yolk, covering about two-thirds of the yolk surface. Separated eggs retain the ring-like shallow depressions that were produced by contact with other eggs.

Fourth day (Fig. 1, No. 4). The blastodisc is reduced to a small opening through which the yolk bulges. The neural keel (future central nervous system) is well defined, encircling almost half of the yolk. The optic vesicles are present but rudimentary.

Fifth day. The three main divisions of the brain are distinguishable. The closing of the blastopore is complete. Three to four myomere blocks are present on either side of the neural keel.

Sixth day (Fig. 1, No. 5). The embryo has increased in length and is well defined. The optic cups can be seen surrounding the spherical lens tissue. Approximately 13 pairs of myomeres are present, arising posterior to the faint outlines of the auditory placodes. Kupffer's vesicle is present as two small sacs beneath the tail bud.

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Seventh day (Fig. 1, No. 6). The lobes of the brain are well defined and from the dorsal aspect the ventricles of the prosencephalon, mesencephalon and the rhombencephalon are conspicuous. Each ventricle is covered by a thin roof. The optic cups enclose the lenses but as yet the chorioid fissure has not closed. The anterior myomeres are chevron-shaped, and the tail bud has begun to lift clear of the yolk.

Ninth day (Fig. 1, Nos. 7 & 8). There is a distinct pericardial cavity beneath, and extending forward of, the head. The heart beats faintly, but there is no visible circulation, and the crimson-purple of the yolk obscures any sign of blood-island formation. The head is expanded laterally, and the brain ventricles, especially that of the mesencephalon, are relatively large. Pigmentation of the chorioid of the eye has begun. The gut is tubular, and there are scattered, stellate melanophores in the trunk muscle directly above it.

Eleventh day (Fig. 1, No. 9). The heart beats regularly and strongly, and a flow of blood through the dorsal and ventral blood vessels is obvious. The sinus venosus is pronounced, lying within the pericardial cavity slightly forward of the head, and receiving blood from the large vitelline vessels. The chorioid fissure has closed completely and appears as a faint white line on the ventral aspect of the eye. Chorioid pigmentation has increased. The brain lobes are more rounded and two otoliths are present in each otic vesicle. The tail bud is less rounded and extends well clear of the yolk, and the upper parietal peritoneum is covered by scattered melanophores. A single large oil globule is present in the yolk.

Fourteenth day (Fig. 1, No. 10). The head is broad and has lifted from the yolk, exposing the chambers of the heart and the rudimentary lower jaw. The eyes are prominent and the lenses can still be seen through the chorioid pigment. Pectoral fin buds are present and the tail is turned to lie parallel with the body.

Sixteenth day (Fig. 1, No. 11). The external olfactory pits appear as shallow depressions anterior to the telencephalon. The tail overlaps the head and there is an increase in the peritoneal and segmental pigmentation. The peritoneal melanophores extend forward beneath the myelencephalon. The embryo changes position frequently.

Twenty-fourth day (Fig. 1, No. 12). The yolk is reduced considerably and the ventral aspect of the embryo faces upwards. Pigmentation of the eye appears complete with many small iridiophores present. The embryo is cramped within the chorion and the tail completely overlaps the head and turns on itself. The mouth, olfactory bulbs and pectoral fins are well formed. The gut is large and convoluted. The liver lies posterior to the yolk and contains a green spherical gallbladder. Just prior to hatching the embryo becomes agitated and the tail begins to flex. As a result, the chorion is ruptured and the prolarva is released.

Prolarva (Fig. 2, Nos. 1 & 2). Prolarval length on hatching ranges from 5.00mm - 6.05mm s.l. There is considerable variation in the amount of yolk present in each prolarva immediately after hatching. Large amounts of yolk in some prolarvae may be accounted for by premature hatching, induced by disturbance. The yolk contains a single reduced oil globule. The heart is prominent and lies on the anterior yolk margin. The gut is long and convoluted and extends beyond the mid-length of the body. Numerous melanophores (30-50) are present on the myomeres page 5of the body and tail, the first of which is placed behind the pectoral fin base; this series extends to the fifth myomere past the vent. Occasional specimens have as few as 6 melanophores on the myomeres. A yellow tinge is present deep in the muscle tissue directly beneath the melanophores of the body and tail. Numerous melanophores line the upper parietal peritoneum. The head is broad and blunt, and there are two clusters of stellate melanophores beneath the myelencephalon. The prolarvae are positively phototropic.

Eight day larva (Fig. 2, Nos. 3 & 4). 6.20mm s.l. The yolk is almost completely absorbed and the oil globule is very small. Pigmentation is unaltered except for the development of several melanophores at the base of each pectoral fin. A yellow tinge surrounds the base of the brain, particularly the myelencephalon, and extends posteriorly within the myomeres above the gut.

Fifteenth day larva (Fig. 2, Nos. 5 & 6). 7.70mm s.l. The overall shape of the larva has altered slightly. There is an increase in the depth of the tail and the relative size of the gut. The pigment pattern is unchanged. Sucker buds are visible at the base of the pectoral fin. Myomeres have the double chevron pattern and the notochord is slightly upturned in the tail. Six rudimentary caudal rays are present. Larval and prolarval measurements in millimetres are as follows:

	Prolarvae	10 days	15 days
Number of Fish	25	25	1
Total length (mm)	5.67(5.40-6.60)	7.12(6.95-7.30)	7.90
Standard length (mm)	5.28(5.00-6.05)	6.70(6.55-6.90)	7.70
Head length (mm)	1.10(1.00-1.15)	1.30(1.25-1.40)	1.70
Eye length (mm)	0.52(0.50-0.54)	0.54(0.51-0.56)	0.65
Snout to vent (mm)	3.32(3.20-3.51)	4.41(4.15-4.55)	5.00
Greatest depth (mm)	1.15(1.00-1.21)	1.02(0.90-1.10)	1.35

Spawning and Development of T. Pinnulatus

In the Lyall Bay area T. pinnulatus begins spawning early in July and continues through to mid-October. South of Cook Strait spawning occurs later. Along the Canterbury coast north of Christchurch the "Earliest records of spawning in the field were made late in August in both 1962 and 1963" (Coakley 1964). Egg masses are found beneath large and small stones, Haliotis shells and other debris which offer secure shelter and remain covered with water at low tide. The egg mass from which this description is made was small (4 × 1.5cm) and contained 198 eggs. Typically, however, egg masses are large (up to 10 × 7cm) and contain as many as 1500 eggs. Coakley (1964) observed that the earliest spent females were the largest, indicating that the larger fish spawn early in the season.

The eggs are laid close together forming flat, irregular-shaped masses. The larger egg clusters always contain groups of eggs which differ appreciably in colour, that is white, orange, pale pink, crimson-red, or pale yellow. There is little intergrading of these colours. Egg sub-groups differing in colour in this way may be at similar stages of development. The variation in colour, unlike that of D. puniceus, does not represent the progressive depletion of the yolk supply in the eggs as development advances, although as time proceeds there is a paling of page 6the colour of each sub-group. It suggests, rather, that the different coloured sub-groups are laid on the same substrate by different females, and that the variation in yolk colour is due to some biochemical difference between each contributing female.

The male T. pinnulatus found with the eggs in the aquarium remained close to them even when alternative shelter was provided. It lay over the eggs, fanning the pectoral and caudal fins, thus maintaining a constant flow of water over the surface of the egg cluster. This current again helped to prevent the accumulation of detritus on the egg membrane. Well over 90% of the egg masses observed on the shore had an adult T. pinnulatus in attendance. Coakley (1964) showed, by dissection, that the majority of adults found close to the egg clusters were males, but only one attendant adult was sexed in the present study.

Development of the Egg and Prolarva

The eggs are oval (mean dimensions of a standard sample of 100 are 1.81 × 1.48mm), dorsoventrally depressed and attached to the substrate by a flattened adhesive base. At an early stage of development the yolk is central and subspherical and has a mean diameter of 1.30mm. Development to hatching (Fig. 3, Nos. 1-11) takes 24 days at 11.5°C. No eggs were found earlier than the gastrulation stage, and these were estimated to be approximately two days old.

Second day (Fig. 3, No. 1). The blastodisc has spread half way around the yolk. Epiboly is not obvious. There are six main oil globules and about 150 smaller ones present in the yolk.

Third day (Fig. 3, No. 2). The blastodisc continues to expand over the yolk, covering about two-thirds of the yolk surface. The embryonic shield is present as a thickened strip of the blastodisc.

Fourth day. The blastopore is reduced to a small opening bordered by the thickened germ ring. The narrow embryonic shield lies notched into the yolk. Many of the small oil globules have coalesced to form large globules.

Fifth day (Fig. 3, No. 3). The closing of the blastopore is complete. The optic vesicles are present but rudimentary. The embryo is still deeply notched into the yolk, particularly in the head region, and the tail is flat.

Seventh day (Fig. 3, No. 4). The embryo has increased in length, encircling more than half of the yolk. There is a distinct pericardial cavity anterior to the head. The three main divisions of the brain are distinguishable. The lens tissue is separate from the ectoderm. Approximately 11 pairs of myomeres are present, arising posterior to the faint outlines of the auditory placodes. Kupffer's vesicle lies beneath the developing tail bud.

Eighth day (Fig. 3, Nos. 5 & 6). The brain, from the dorsal aspect, shows clearly defined ventricles, each covered by a thin roof. The optic cups surround the spherical lens tissue, and the chorioid fissure has not yet closed. The anterior myomeres are chevron-shaped, and the tail bud begins to lift clear of the yolk.

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Tenth day (Fig. 3, No. 7). The heartbeat is regular, sending blood slowly through the major blood vessels. The head has expanded laterally, and the brain ventricles, especially that of mesencephalon, have increased in size. The chorioid is slightly pigmented and the fissure has closed. Two small otoliths are present in each round otic vesicle. The gut is thin and tubular.

Fourteenth day. The head is broad and has lifted from the yolk, exposing the chambers of the heart and the developing lower jaw. The sinus venosus is a thin expanded sac lying within the pericardial cavity forward of the head, and receiving blood from the large vitelline vessels. The brain lobes are rounded and larger. The chorioid fissure appears as a faint white line on the ventral aspect of the eye. The eyes are prominent and the lenses can still be seen through the chorioid pigment. The upper parietal peritoneum has scattered melanophores. Pectoral fin buds are present and the tail has turned to lie parallel with the head and trunk. A single large oil globule and ten smaller ones are present in the yolk.

Sixteenth day (Fig. 3, No. 8). Very little change has taken place, except that the jaws appear more definite and slightly fleshy.

Twentieth day (Fig. 3, Nos. 9 & 10). The olfactory pits appear as shallow depressions anterior to the telencephalon. The tail overlaps the head and there is an increase in the number of peritoneal melanophores. The embryo alters position frequently. A single large oil globule remains in the yolk.

Twenty-fourth day (Fig. 3, No. 11). The yolk is reduced considerably and the ventral aspect of the embryo faces upwards. Pigmentation of the chorioid appears complete, the silvery appearance of the eye being due to the presence of iridiophores. The embryo is cramped within the egg and the tail completely overlaps the head and turns on itself. The mouth, olfactory bulbs and pectoral fins are well formed. The gut is long and convoluted. The liver and the green spherical gallbladder lie behind the yolk. The upper peritoneal pigment is arranged approximately as two rows of 10 large stellate melanophores, extending from above the liver to the vent. In some individuals two rows of 3-4 stellate melanophores are present on the lower parietal peritoneum extending back from the yolk. Just prior to hatching the embryo becomes active and begins to flex its tail. As a result, the chorion is ruptured and the prolarva is released.

Prolarva (Fig. 4, Nos. 1 and 2). Prolarval length on hatching ranges from 5.35mm - 6.10mm. Like D. puniceus there was some variation in the amount of yolk present in each prolarva immediately after hatching. The yolk contains a single reduced oil globule, and the heart lies on the anterior margin of the yolk sac. The gut is long and convoluted and extends past the mid-length of the body. Upper peritoneal pigmentation is scattered and extends from above the liver to the vent. Some individuals retain the lower peritoneal pigmentation. A yellow tinge lies within the muscles directly above the peritoneal pigment, and extends beneath the otic capsules and the hind-brain. Two stellate melanophores are present beneath each pectoral fins, but no pigment is present on the head.

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The measurements of prolarvae are as follows:

Number of fish	23
Standard length (mm)	5.70 (5.35-6.10)
Head length (mm)	1.04 (0.95-1.10)
Eye length (mm)	0.43 (0.40-0.45)
Snout to vent (mm)	3.43 (3.20-3.60)
Greatest depth (mm)	1.02 (0.80-1.20)